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[Preprint]. 2025 Jul 16:2025.07.13.664599. [Version 1] doi: 10.1101/2025.07.13.664599

Gene model for the ortholog of tgo in Drosophila pseudoobscura

Megan E Lawson 1, Janelle Marie Suriaga 2, Mary B Rowland 1, Laura K Reed 1, Chinmay P Rele 1, Jacqueline K Wittke-Thompson 2,Ω
PMCID: PMC12338613  PMID: 40791386

Abstract

Gene model for the ortholog of tango (tgo) in the April 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Introduction

This article reports a predicted gene model generated by undergraduate work using a structured gene model annotation protocol defined by the Genomics Education Partnership (GEP; thegep.org) for Course-based Undergraduate Research Experience (CURE). The following information in quotes may be repeated in other articles submitted by participants using the same GEP CURE protocol for annotating Drosophila species orthologs of Drosophila melanogaster genes in the insulin signaling pathway.

“In this GEP CURE protocol students use web-based tools to manually annotate genes in non-model Drosophila species based on orthology to genes in the well-annotated model organism fruit fly Drosophila melanogaster. The GEP uses web-based tools to allow undergraduates to participate in course-based research by generating manual annotations of genes in non-model species (Rele et al., 2023). Computational-based gene predictions in any organism are often improved by careful manual annotation and curation, allowing for more accurate analyses of gene and genome evolution (Mudge and Harrow 2016; Tello-Ruiz et al., 2019). These models of orthologous genes across species, such as the one presented here, then provide a reliable basis for further evolutionary genomic analyses when made available to the scientific community.” (Myers et al., 2024).

“The particular gene ortholog described here was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila. The Insulin/insulin-like growth factor signaling pathway (IIS) is a highly conserved signaling pathway in animals and is central to mediating organismal responses to nutrients (Hietakangas and Cohen 2009; Grewal 2009).” (Myers et al., 2024).

“The Drosophila tango (CG11987, FBgn0264075) gene encodes a bHLH-PAS protein that controls CNS midline and tracheal development (Sonnenfeld et al., 1997). Both cell culture and in vivo studies have shown that a DNA enhancer element acts as a binding site for both Single- minded::Tango and Trachealess::Tango heterodimers and functions in controlling CNS midline and tracheal transcription. Isolation and analysis of tango mutants reveal CNS midline and tracheal defects. In addition, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-α and HIF-β homologues, respectively, in a conserved hypoxia-inducible transcriptional response in Drosophila melanogaster that is homologous to the mammalian HIF-dependent response (Lavista-Llanos et al., 2002). Insulin has been shown to activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos, and this effect is mediated by PI3K-AKT and TOR pathways. Overexpression of dAKT and dPDK1 in normoxic embryos provoked a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment (Dekanty et al., 2005).” (Lawson 2025a).

“D. pseudoobscura is part of the pseudoobscura species subgroup within the obscura species group in the subgenus Sophophora of the genus Drosophila (Sturtevant 1942; Buzzati-Traverso & Scossiroli 1955). It was first described by Frolowa (1929). The pseudoobscura species subgroup is endemic to the New World, where D. pseudoobscura is distributed throughout Western North American, and south through Mexico and Central America (Markow & O’Grady 2005). An additional population of D. pseudoobscura, found near Bogota, Columbia, is partially reproductively isolated from the North and Central American populations (Prakash 1972). It is found primarily in chaparral and temperate forests. D. pseudoobscura has been studied extensively in the context of ecological and behavioral genetics, speciation, and genome evolution (Powell 1997; Lawson et al., 2024).” (Lawson 2025b).

We propose a gene model for the ortholog in D. pseudoobscura (NCBI taxon ID 46245) of the D. melanogaster tango (tgo) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_001359833.1 (Locus ID LOC4803031) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly of D. pseudoobscura (GCA_000001765.2, Chen et al., 2014). This model is based on RNA-Seq data from D. pseudoobscura (SRP006203) and tgo in D. melanogaster using FlyBase release FB2024_02 (GCA_000001215.4; Gramates et al., 2022; Jenkins et al., 2022; Larkin et al., 2021).

Synteny

The target gene, tgo, occurs on chromosome 3R in D. melanogaster and is flanked upstream by neuralized (neur) and hyrax (hyx) and downstream by CG11986 and Splicing factor 3b subunit 5 (Sf3b5). The tblastn search of D. melanogaster tgo-PA (query) against the D. pseudoobscura (GenBank Accession: GCA_000001765.2 Genome Assembly (database) placed the putative ortholog of tgo within scaffold CM000070 (CM000070.4) at locus LOC4803031 (XP_001359833.1)— with an E-value of 0.0 and a percent identity of 83.05%. Furthermore, the putative ortholog is flanked upstream by LOC4803030 (XP_001359832.3) and LOC4803029 (XP_001359831.1), which correspond to neur and hyx in D. melanogaster (E-value: 0.0 and 0.0; identity: 86.62% and 91.48%, respectively, as determined by blastp; Figure 1A; Altschul et al., 1990). The putative ortholog of tgo is flanked downstream by LOC4803033 (XP_033232624.1) and LOC4803032 (XP_015038250.1), which correspond to CG11986 and Kcmf1 in D. melanogaster (E-value: 0.0 and 0.0; identity: 81.39% and 81.19%, respectively, as determined by blastp). The putative ortholog assignment for tgo in D. pseudoobscura is supported by the following evidence: Most of the genes surrounding the tgo ortholog are orthologous to the genes at the same locus in D. melanogaster and local synteny is mostly conserved aside from the second most downstream gene, supported by E-values and percent identities, so we conclude that LOC4803031 is the correct ortholog of tgo in D. pseudoobscura (Figure 1A).

Figure 1. tgo gene model comparison between Drosophila pseudoobscura and Drosophila melanogaster orthologs.

Figure 1.

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(A) Synteny comparison of the genomic neighborhoods for tgo in Drosophila melanogaster and D. pseudoobscura. Thin underlying arrows indicate the DNA strand within which the target gene–tgo–is located in D. melanogaster (top) and D. pseudoobscura (bottom). The thin arrow pointing to the left indicates that tgo is on the negative (−) strand in D. melanogaster, and the thin arrow pointing to the right indicates that tgo is on the positive (+) strand in D. pseudoobscura. The wide gene arrows pointing in the same direction as tgo are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of tgo are on the opposite strand relative to the thin underlying arrows. White gene arrows in D. pseudoobscura indicate orthology to the corresponding gene in D. melanogaster, while black gene arrows indicate non-orthology. Gene symbols given in the D. pseudoobscura gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. pseudoobscura. (B) Gene Model in GEP UCSC Track Data Hub (Raney et al., 2014). The coding-regions of tgo in D. pseudoobscura are displayed in the User Supplied Track (black); coding exons are depicted by thick rectangles and introns by thin lines with arrows indicating the direction of transcription. Subsequent evidence tracks include BLAT Alignments of NCBI RefSeq Genes (dark blue, alignment of Ref-Seq genes for D. pseudoobscura), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), Transcripts and Coding Regions Predicted by TransDecoder (dark green), RNA-Seq from Adult Females and Adult Males (red and light blue, respectively; alignment of Illumina RNA-Seq reads from D. pseudoobscura), and Splice Junctions Predicted by regtools using D. pseudoobscura RNA-Seq (SRP006203). Splice junctions shown have a minimum read-depth of 10 with 10–49 supporting reads indicated in blue. (C) Dot Plot of tgo-PA in D. melanogaster (x-axis) vs. the orthologous peptide in D. pseudoobscura (y-axis). Amino acid number is indicated along the left and bottom; coding-exon number is indicated along the top and right, and exons are also highlighted with alternating colors. Line breaks in the dot plot indicate mismatching amino acids at the specified location between species. The boxed region highlights a tandem repeat within the amino acid sequence.

Protein Model

tgo in D. pseudoobscura has two identical protein-coding isoforms (tgo-PA and tgo-PB; Figure 1B). These isoforms (tgo-PA and tgo-PB) contain one protein-coding exon. Relative to the ortholog in D. melanogaster, the coding-exon number and isoform count are conserved, as tgo in D. melanogaster also has two identical isoforms, each with one exon. The sequence of tgo-PA in D. pseudoobscura has 94.92% identity (E-value: 0.0 with the protein-coding isoform tgo-PA in D. melanogaster, as determined by blastp (Figure 1C). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accessions BK065289 and BK065290). This gene model can also be seen within the target genome at this TrackHub.

Methods

“Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (2023). Briefly, students use the GEP instance of the UCSC Genome Browser v.435 (https://gander.wustl.edu; Kent WJ et al., 2002; Navarro Gonzalez et al., 2021) to examine the genomic neighborhood of their reference IIS gene in the D. melanogaster genome assembly (Aug. 2014; BDGP Release 6 + ISO1 MT/dm6). Students then retrieve the protein sequence for the D. melanogaster reference gene for a given isoform and run it using tblastn against their target Drosophila species genome assembly on the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al., 1990) to identify potential orthologs. To validate the potential ortholog, students compare the local genomic neighborhood of their potential ortholog with the genomic neighborhood of their reference gene in D. melanogaster. This local synteny analysis includes at minimum the two upstream and downstream genes relative to their putative ortholog. They also explore other sets of genomic evidence using multiple alignment tracks in the Genome Browser, including BLAT alignments of RefSeq Genes, Spaln alignment of D. melanogaster proteins, multiple gene prediction tracks (e.g., GeMoMa, Geneid, Augustus), and modENCODE RNA-Seq from the target species. Detailed explanation of how these lines of genomic evidenced are leveraged by students in gene model development are described in Rele et al. (2023). Genomic structure information (e.g., CDSs, intron-exon number and boundaries, number of isoforms) for the D. melanogaster reference gene is retrieved through the Gene Record Finder (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html; Rele et al., 2023). Approximate splice sites within the target gene are determined using tblastn using the CDSs from the D. melanogaster reference gene. Coordinates of CDSs are then refined by examining aligned modENCODE RNA-Seq data, and by applying paradigms of molecular biology such as identifying canonical splice site sequences and ensuring the maintenance of an open reading frame across hypothesized splice sites. Students then confirm the biological validity of their target gene model using the Gene Model Checker (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html; Rele et al., 2023), which compares the structure and translated sequence from their hypothesized target gene model against the D. melanogaster reference gene model. At least two independent models for a gene are generated by students under mentorship of their faculty course instructors. Those models are then reconciled by a third independent researcher mentored by the project leaders to produce the final model. Note: comparison of 5’ and 3’ UTR sequence information is not included in this GEP CURE protocol.” (Gruys et al., 2025)

Supplementary Material

Supplement 1
media-1.zip (6.4KB, zip)

Supplemental Files

1. Zip file containing a FASTA, PEP, GFF files for the gene model

2. Figure 1 in high resolution

Acknowledgements

We would like to thank Wilson Leung for developing and maintaining the technological infrastructure that was used to create this gene model and Laura K. Reed for overseeing the project. Thank you to FlyBase for providing the definitive database for Drosophila melanogaster gene models. Further, we would like to thank the editors and developers at the journal microPublication: Biology for assistance in developing the template for these single gene ortholog publications.

Funding

This material is based upon work supported by the National Science Foundation (1915544) and the National Institute of General Medical Sciences of the National Institutes of Health (R25GM130517) to the Genomics Education Partnership (GEP; https://thegep.org/; PI-LKR). Any opinions, findings, and conclusions or recommendations expressed in this material are solely those of the author(s) and do not necessarily reflect the official views of the National Science Foundation nor the National Institutes of Health.

Footnotes

Metadata: Bioinformatics, Genomics, Drosophila, Genotype Data, New Finding

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1
media-1.zip (6.4KB, zip)

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